Hyaluronan and cardiac regeneration.

Hyaluronan (HA) is abundantly expressed in several human tissues and a variety of roles for HA has been highlighted. Particularly relevant for tissue repair, HA is actively produced during tissue injury, as widely evidenced in wound healing investigations. In the heart HA is involved in physiological functions, such as cardiac development during embryogenesis, and in pathological conditions including atherosclerosis and myocardial infarction. Moreover, owing to its relevant biological properties, HA has been widely used as a biomaterial for heart regeneration after a myocardial infarction. Indeed, HA and its derivatives are biodegradable and biocompatible, promote faster healing of injured tissues, and support cells in relevant processes including survival, proliferation, and differentiation. Injectable HA-based therapies for cardiovascular disease are gaining growing attention because of the benefits obtained in preclinical models of myocardial infarction. HA-based hydrogels, especially as a vehicle for stem cells, have been demonstrated to improve the process of cardiac repair by stimulating angiogenesis, reducing inflammation, and supporting local and grafted cells in their reparative functions. Solid-state HA-based scaffolds have been also investigated to produce constructs hosting mesenchymal stem cells or endothelial progenitor cells to be transplanted onto the infarcted surface of the heart. Finally, applying an ex-vivo mechanical stretching, stem cells grown in HA-based 3D scaffolds can further increase extracellular matrix production and proneness to differentiate into muscle phenotypes, thus suggesting a potential strategy to create a suitable engineered myocardial tissue for cardiac regeneration.

Restored perfusion and reduced inflammation in the infarcted heart after grafting stem cells with a hyaluronan-based scaffold.

The aim of this study is to investigate the blood perfusion and the inflammatory response of the myocardial infarct area after transplanting a hyaluronan-based scaffold (HYAFF(®) 11) with bone marrow mesenchymal stem cells (MSCs). Nine-week-old female pigs were subjected to a permanent left anterior descending coronary artery ligation for 4 weeks. According to the kind of the graft, the swine subjected to myocardial infarction were divided into the HYAFF(®) 11, MSCs, HYAFF(®) 11/MSCs and untreated groups. The animals were killed 8 weeks after coronary ligation. Scar perfusion, evaluated by Contrast Enhanced Ultrasound echography, was doubled in the HYAFF(®) 11/MSCs group and was comparable with the perfusion of the healthy, non-infarcted hearts. The inflammation score of the MSCs and HYAFF(®) 11/MSCs groups was near null, revealing the role of the grafted MSCs in attenuating the cell infiltration, but not the foreign reaction strictly localized around the fibres of the scaffold. Apart from the inflammatory response, the native tissue positively interacted with the HYAFF(®) 11/MSCs construct modifying the extracellular matrix with a reduced presence of collagene and increased amount of proteoglycans. The border-zone cardiomyocytes also reacted favourably to the graft as a lower degree of cellular damage was found. This study demonstrates that the transplantation in the myocardial infarct area of autologous MSCs supported by a hyaluronan-based scaffold restores blood perfusion and almost completely abolishes the inflammatory process following an infarction. These beneficial effects are superior to those obtained after grafting only the scaffold or MSCs, suggesting that a synergic action was achieved using the cell-integrated polymer construct.

Comparison between stem cells harvested from wet and dry lipoaspirates.

Adipose-derived stem cells (ASC) are usually isolated from lipoaspirates, but it is not known if the anesthetic solution injected into adipose tissue affects cell yield and functions. Two different samples were drawn from the abdominal region of female subjects. In the first, a physiological solution containing lidocaine/adrenaline was injected (wet liposuction, WL), while in the contralateral area, the sample was collected without injecting any solution (dry liposuction, DL). The aspirates were processed to investigate the yield of the stromal-vascular fraction (SVF) cells and ASC frequency, growth rate, apoptosis, and differentiation potential. The solid dried mass of fresh WL isolates was lower than that of DL isolates (p < 0.01) due to the presence, in the former, of a liquid solution. As a consequence, the amount of WL-SVF cells was 18.7% lower than those obtained from DL (p < 0.01); this difference was also observed under culture conditions. In addition, the number of colony-forming unit-fibroblasts (CFU-Fs) obtained from 1 × 10(3) SVF cells was 25.5% lower in WL-aspirates than DL-aspirates (p < 0.05) owing, at least in part, to the observed presence of ASC [corrected] in the liquid solution of the WL isolates. After WL and DL, no differences were observed in ASC growth rate, apoptosis, or differentiation potential toward adipogenic, osteogenic, and endothelial cell lineages. In conclusion, WL yields about 40% fewer ASC than DL due to the combined effect of tissue dilution and the reduced frequency of ASC in the SVF. The main biological features of ASC are suitable for cell-based therapies.

A pro-survival effect of polyamine depletion on norepinephrine-mediated apoptosis in cardiac cells: role of signaling enzymes.

Recent studies report that the primary transmitter of sympathetic nervous system norepinephrine (NE), which is actively produced in failing human heart, is able to induce apoptosis of rat cardiomyocytes. Apoptotic cell death of cardiomyocytes is involved in several cardiovascular diseases including ischemia, hypertrophy and heart failure, therefore representing a potential therapeutic target. The natural occurring polyamines, putrescine, spermidine and spermine, are biogenic amines involved in many cellular processes, including apoptosis. Thus, we have studied the involvement of polyamines in the apoptosis of cardiac cells induced by the treatment with NE. The results indicate that NE caused an early induction of the activity of ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis, followed by a later increase of apoptotic cell death. This effect was prevented in the presence of α-difluoromethylornithine, an irreversible inhibitor of ODC. Moreover, the study of some key signal transduction pathways revealed an involvement of AMP-activated protein kinase, AKT and p38 mitogen-activated protein kinases, in the modulation by polyamines of the response of cardiomyocytes to NE. In fact, polyamine-depleted cells showed an altered activation pattern of these kinases that may contrast apoptosis and appeared to result from a differential effect on the specific phosphatases that dephosphorylate and switch off these signaling proteins. In conclusion, these results indicate that in cardiac cells polyamines are involved in the execution of the death program activated by NE, and suggest that their apoptosis facilitating action is mediated by a network of specific phosphatases and kinases.

Sharing pathogenetic mechanisms between acute myocardial infarction and Alzheimer's disease as shown by partially overlapping of gene variant profiles.

Gene variants that promote inflammation and cholesterol metabolism have been associated with acute myocardial infarction (AMI) and Alzheimer's disease (AD). We investigated a panel of relevant polymorphisms to distinguish genetic backgrounds for AMI and AD: IL10 -1082G/A, IL6 -174G/C, TNF -308G/A, IFNG +874T/A, SERPINA3 -51G/T, HMGCR -911C/A, APOE ε2/3/4 (280 AMI cases, 257 AD cases, and 1307 population controls, all Italian (presumed risk alleles are shown in bold). Six genetic risk sets I to VI were identified by fuzzy latent classification: I had low risk; II and III had low risk before age 65 (II, III); low risk sets lacked pro-inflammatory alleles for HMGCR-TNF-APOE. Pro-inflammatory alleles for SERPINA3-IL10-IFNG were found for high risk sets IV to VI. Set IV 'AMI < age 40, AD < age 65' included risk alleles for HMGCR. Set V 'AMI over a broad range of age' included risk alleles for TNF+IL6. Set VI 'AMI at ages 40 to 55, AD ages 65+' included APOE ε4. Close resemblance to the high risk sets, as indicated by membership scores close to one, defined high relative risks. We conclude that AMI and AD share genetic backgrounds involving cholesterol metabolism and the upregulation of inflammation and that gene-gene interactions in relevant sets of genes may be useful in defining inherited risk for common disorders.

Leupeptin preserves cardiac nitric oxide synthase 3 during reperfusion following long-term cardioplegia.

The objective of this study was to investigate how long-term cardioplegia/reperfusion affects cardiac nitric oxide synthase 3 (NOS3). To this aim, rat hearts were mounted in a perfusion apparatus and equilibrated with a modified Krebs-Henseleit solution (KH). The hearts were then arrested by soaking them in cold St. Thomas Hospital II solution (STH) for 5, 7, and 15 h. Reperfusion was performed by low-flow cold STH delivering for 1 h followed by 15-min aerobic normothermic KH perfusion. Cardioplegia preserved the amount of NOS3 irrespective of the duration of the cardiac arrest. NOS3 content was also unaffected by reperfusion following 5 and 7 h of cardioplegia. On the contrary, reperfusion performed after 15 h of cardioplegia caused a marked reduction in the amount of NOS3 protein, in both endothelial and cardiac muscle cells, and NOS activity. The involvement of intracellular proteolysis as a cause of reduction in NOS3 cardiac level was then investigated by delivering 0.1 mmol/L of either calpain I and II inhibitors or 0.05 mmol/L leupeptin during heart reperfusion. Only the treatment with leupeptin preserved NOS3, indicating that lysosomal proteases rather then cytoplasmic calpains were mainly responsible for the cleavage of this enzyme. The observed decrease in GSH/GSSG ratio and activation of JNK in the reperfused heart suggested that proteolysis could be triggered by reactive oxygen species.

Comparison between Culture Conditions Improving Growth and Differentiation of Blood and Bone Marrow Cells Committed to the Endothelial Cell Lineage.

The aim of this study was to compare different cell sources and culture conditions to obtain endothelial progenitor cells (EPCs) with predictable antigen pattern, proliferation potential and in vitro vasculogenesis. Pig mononuclear cells were isolated from blood (PBMCs) and bone marrow (BMMCs). Mesenchymal stem cells (MSCs) were also derived from pig bone marrow. Cells were cultured on fibronectin in the presence of a high concentration of VEGF and low IGF-1 and FGF-2 levels, or on gelatin with a lower amount of VEGF and higher IGF-1 and FGF-2 concentrations. Endothelial commitment was relieved in almost all PBMCs and BMMCs irrespective of the protocol used, whilst MSCs did not express a reliable pattern of EPC markers under these conditions. BMMCs were more prone to expand on gelatin and showed a better viability than PBMCs. Moreover, about 90% of the BMMCs pre-cultured on gelatin could adhere to a hyaluronan-based scaffold and proliferate on it up to 3 days. Pre-treatment of BMMCs on fibronectin generated well-shaped tubular structures on Matrigel, whilst BMMCs exposed to the gelatin culture condition were less prone to form vessel-like structures. MSCs formed rough tubule-like structures, irrespective of the differentiating condition used. In a relative short time, pig BMMCs could be expanded on gelatin better than PBMCs, in the presence of a low amount of VEGF. BMMCs could better specialize for capillary formation in the presence of fibronectin and an elevated concentration of VEGF, whilst pig MSCs anyway showed a limited capability to differentiate into the endothelial cell lineage.

Architectural organization and functional features of early endothelial progenitor cells cultured in a hyaluronan-based polymer scaffold.

Neovascularization can be improved using polymer scaffolds supporting endothelial progenitor cells (EPCs). The aim of the present study was to investigate whether human early EPCs (eEPCs) could be efficiently cultured in a hyaluronan-based non-woven mesh (HYAFF-11). eEPCs were seeded on HYAFF-11 at the density of 1 x 10(6)/cm(2) and cultured with endothelial differentiating factors for 3 weeks. After 24 h, nearly 90% of EPCs were adherent. Cell viability, evaluated by methyltetrazolium test, was greater in HYAFF-11 than on the most commonly used fibronectin-coated dishes, even if a progressive decline in viability was observed starting from approximately the second week of culture. eEPCs easily migrated to and aggregated on the scaffold. Evidence of active protein synthesis and features of endothelial differentiation, including cellular transcytotic channels and micropinocytotic vesicles, was revealed using electron microscopy, immunofluorescence, and reverse transcriptase polymerase chain reaction analysis. eEPCs cultured in the scaffold also showed a certain angiogenic activity, as demonstrated by hepatocyte growth factor transcription and vascular endothelial growth factor secretion. In conclusion, eEPCs can migrate and adhere inside HYAFF-11, maintain their pre-endothelial phenotype, and express angiogenic factors, especially within the first week of growth. These results indicate that non-woven HYAFF-11 could be a promising candidate as a vehicle for eEPCs for regenerative medicine applications.

Difluoromethylornithine stimulates early cardiac commitment of mesenchymal stem cells in a model of mixed culture with cardiomyocytes.

The efficiency of in vitro mesenchymal stem cell (MSC) differentiation into the myocardial lineage is generally poor. In order to improve cardiac commitment, bone marrow GFP+MSCs obtained from transgenic rats were cultured with adult wild type rat cardiomyocytes for 5 days in the presence of difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis and cell proliferation. The percentage of GFP+MSCs showing cardiac myofibril proteins (cMLC2, cTnI) was about threefold higher after DFMO addition (3%) relative to the untreated control (1%). Another set of experiments was performed with cardiomyocytes incubated for 1 day in the absence of glucose and serum and under hypoxic conditions (pO2 < 1%), in order to simulate severe ischemia. The percentage of cardiac committed GFP+MSCs was about 5% when cultured with the hypoxic/starved cardiomyocytes and further increased to 7% after DFMO addition. The contemporary presence of putrescine in DFMO-treated cells markedly blunted differentiation, while the cytostatic mitomycin C was not able to induce cardiac commitment. The involvement of histone acetylation in DFMO-induced differentiation was evidenced by the strong attenuation of cardiac commitment exerted by anacardic acid, an inhibitor of histone acetylase. Moreover, the percentage of acetylated histone H3 significantly increased in bone marrow MSCs obtained from wild type rats and treated with DFMO. These results suggest that polyamine depletion can represent a useful strategy to improve MSC differentiation into the cardiac lineage, especially in the presence of cardiomyocytes damaged by an ischemic environment.

Effect of the polyamine analogue N1,N11-diethylnorspermine on cell survival and susceptibility to apoptosis of human chondrocytes.

Chondrocyte survival is closely linked to cartilage integrity, and forms of chondrocyte apoptotic death can contribute to cartilage degeneration in articular diseases. Since growing evidence also implicates polyamines in the control of cell death, we have been investigating the role of polyamine metabolism in chondrocyte survival and apoptosis. Treatment of human C-28/I2 chondrocytes with N(1),N(11)-diethylnorspermine (DENSPM), a polyamine analogue with clinical relevance as an experimental anticancer agent, inhibited polyamine biosynthesis and induced polyamine catabolism, thus rapidly depleting all main polyamines. DENSPM did not increase significantly caspase activity, but provoked a late cell death associated to DNA fragmentation. A short treatment with DENSPM did not reduce cell viability when given alone, but enhanced caspase-3 and -9 activation in chondrocytes exposed to tumor necrosis factor-alpha (TNF) and cycloheximide (CHX). A longer treatment with DENSPM however reduced caspase response to TNF plus CHX. Depletion of all polyamines obtained by specific inhibitors of polyamine biosynthesis did not cause cell death and contrasted apoptosis by decreasing caspase activities. In conclusion, following DENSPM treatment, C-28/I2 chondrocytes are initially sensitized to caspase 9-dependent apoptosis in the presence of TNF and CHX and may eventually undergo a late and mainly caspase-independent cell death in the absence of other stimuli. Moreover, these results indicate that a reduction of polyamine levels not only leads to inhibition of cell proliferation, but also of caspase-mediated pathways of chondrocyte apoptosis.

Acute myocardial infarction and proinflammatory gene variants.

We identified four genetic risk sets for acute myocardial infarction (AMI) from information on functional gene variants that favor inflammation or modulate cholesterol metabolism: IL6 -174 G/C, TNF -308 G/A, IL10 -1082 G/A, SERPINA3 -51 G/T, IFNG +874 T/A, HMGCR -911 C/A, and APOE epsilon2/3/4; 316 patients and 461 healthy subjects, all Italian. Putative risk alleles are shown underlined. The sets were identified using grade-of-membership analysis. Membership scores in the sets are automatically generated for individuals. The 'low intrinsic risk' set had alleles that downregulate inflammation and cholesterol synthesis (IL6, TNF, ILl0, HMGCR). 'AMI across a broad age range' carried multiple proinflammatory alleles (IL6, TNF, IL10, SERPINA3): All 72 persons like this set were affected yet had relatively low plasma cholesterol levels. 'A subset of AMI in middle age' had numerous proinflammatory alleles (IL6, TNF, SERPINA3, IFNG, HMGCR). 'AMI after age 80' had a reduced risk set (IL6, IL10, IFNG). A total of 95% of cases had >/=50% membership in the high intrinsic risk sets. We conclude that proinflammatory gene variants taken together strongly determine an individual's risk for myocardial infarction. This information may better define the pathogenesis of myocardial infarction and identify individuals who might benefit from early interventions.

Different expression of NOS isoforms in early endothelial progenitor cells derived from peripheral and cord blood.

Cord blood and peripheral-adult blood were compared as different sources of early endothelial precursor cells (eEPCs). Total mononuclear cells (MNCs) were obtained from both blood types and committed to eEPCs by exposure to fibronectin, VEGF, IGF-I, and bFGF. Under this condition, MNCs seeded at the density of 3 x 10(5) cells/cm(2) assumed a spindle shape, which was indicative of developing eEPCs, and expanded in a similar manner irrespective to the blood sources. Ulex europaeus agglutinin (UEA-1) and acetylated low density lipoprotein (acLDL) double staining was present in 90% in both peripheral- and cord-blood eEPCs after 2-week expansion. Also, the ability of eEPCs to form tubule-like structures in Matrigel was independent of their blood source, but dependent on the presence of human umbilical vein endothelial cells (HUVECs). eNOS and nNOS were not detectable by Western blotting in both peripheral and cord-blood eEPCs upon 3 weeks and their mRNA levels were lower than 2% relative to those present in HUVECs. On the contrary, iNOS protein was detectable in peripheral-blood eEPCs, but not in cord-blood eEPCs and HUVECs, as well as iNOS mRNA was more concentrated in peripheral-blood eEPCs than in cord-blood eEPCs and HUVECs. These data suggest that: (a) peripheral and cord blood can be considered comparable sources of eEPCs when they are expanded and differentiated in a short-term period; (b) the extremely low expression of constitutive NOS isoforms in the eEPCs of both blood types should markedly reduce their ability to regulate NO-dependent vasorelaxation; (c) the presence of iNOS in peripheral-blood eEPCs could improve the process of vasculogenesis.

Long-term treatment with N-acetylcysteine, but not caloric restriction, protects mesenchymal stem cells of aged rats against tumor necrosis factor-induced death.

The survival of mesenchymal stem cells (MSCs) to tumor necrosis factor alpha (TNFalpha) stimulation was evaluated after a long-term antioxidant treatment, or caloric restriction, in aged rats. MSCs were isolated from bone marrow of 30-month-old rats which orally received N-acetylcysteine in the last 18 months. The necrotic cell death-induced in vitro by TNFalpha, determined by trypan blue exclusion, was markedly attenuated in MSCs obtained from treated vs. control aged rats (percent mean+/-SEM: 10.9+/-2.17 vs. 17.8+/-0.53; p<0.05). Also, the proliferation rate of MSCs from control, but not N-acetylcysteine-treated, aged rats evaluated up to 2 weeks was significantly higher than that of MSCs from younger (4-month-old) rats. No significant effect was observed relative to the parameters investigated when the aged rats were previously subjected to a hypocaloric diet for 18 months. In conclusion, a prolonged supplementation with N-acetylcysteine in rats can increase resistance to necrotic death of MSCs and may also counteract an excessive rate of MSC proliferation.

Ischemic preconditioning preserves proton leakage from mitochondrial membranes but not oxidative phosphorylation during heart reperfusion.

The aim of this study was to evaluate the role of mitochondria in the recovery of cardiac energetics induced by ischaemic preconditioning at reperfusion. Isolated rat hearts were aerobically perfused (control), subjected to global ischaemia and reperfusion (reperfusion), or subjected to 3 brief cycles of ischaemia/reperfusion and then to the protocol of reperfusion (preconditioning). At the end of the perfusion, antimycin A was delivered to the heart for 25 min, to inhibit mitochondrial respiration and stimulate glycolysis. The increased amount of lactate released in the coronary effluent was correlated with the number of viable cells producing this end-product of glycolysis. Preconditioned hearts released 18% more lactate than reperfused hearts (p < 0.05). This result indicates that preconditioning partially preserved cell viability, as was also evidenced by the MTT assay performed on cardiac biopsies. The difference between antimycin A-stimulated and basal lactate concentration, representing the contribution of mitochondria to the overall energetics of cardiac tissue, was also 18% more elevated in the preconditioned hearts than in the reperfused hearts (p < 0.01). The study of the respiratory function of mitochondria isolated at the end of perfusion, showed that preconditioning did not improve the oxygen-dependent production of ATP (state 3 respiration, ADP/O). On the contrary, state 4 respiration, which is related to proton leakage, was 35.0% lower in the preconditioned group than reperfusion group (p < 0.05). Thus, preconditioning ameliorates cardiac energetics by preserving cell death, but without affecting mitochondrial oxidative phosphorylation. Mitochondria can contribute to cell survival by the attenuation of proton leak from inner membrane.

Activation of glucose transport during simulated ischemia in H9c2 cardiac myoblasts is mediated by protein kinase C isoforms.

Glucose transport into cells may be regulated by a variety of conditions, including ischemia. We investigated whether some enzymes frequently involved in the metabolic adaptation to ischemia are also required for glucose transport activation. Ischemia was simulated by incubating during 3 h H9c2 cardiomyoblasts in a serum- and glucose-free medium in hypoxia. Under these conditions 2-deoxy-d-[2,6-(3)H]-glucose uptake was increased (57% above control levels, p<0.0001) consistently with GLUT1 and GLUT4 translocation to sarcolemma. Tyrosine kinases inhibition via tyrphostin had no effect on glucose transport up-regulation induced by simulated ischemia. On the other hand, chelerythrine, a broad range inhibitor of protein kinase C isoforms, and rottlerin, an inhibitor of protein kinase C delta, completely prevented the stimulation of the transport rate. A lower activation of hexose uptake (19%, p<0.001) followed also treatment with Gö6976, an inhibitor of conventional protein kinases C. Finally, PD98059-mediated inhibition of the phosphorylation of ERK 1/2, a downstream mitogen-activated protein kinase (MAPK), only partially reduced the activation of glucose transport induced by simulated ischemia (31%, p<0.01), while SB203580, an inhibitor of p38 MAPK, did not exert any effect. These results indicate that stimulation of protein kinase C delta is strongly related to the up-regulation of glucose transport induced by simulated ischemia in cultured cardiomyoblasts and that conventional protein kinases C and ERK 1/2 are partially involved in the signalling pathways mediating this process.

Polyamine depletion reduces TNFalpha/MG132-induced apoptosis in bone marrow stromal cells.

Polyamines are powerful modulators of both growth and survival in mammalian cells. In this study, we investigated the possibility of attenuating the process of apoptosis in bone marrow stromal cells (BMSCs), which comprise mesenchymal stem cells, by reducing the intracellular levels of polyamines. BMSCs were isolated from rat femurs and expanded for 12 days. At this time, BMSCs were CD34neg, CD45neg, and mostly CD90pos. BMSCs were grown for an additional 2 days in the presence of 1 mM alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, which reduced the content of both putrescine and spermidine by nearly 90%. DFMO treatment progressively slowed down BMSC proliferation, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay, without arresting their growth completely. The effect of polyamine depletion on caspase-3 activity was evaluated in BMSCs after treatment with 500 U/ml tumor necrosis factor-alpha (TNFalpha) and 5 microM MG132, an inhibitor of proteasome. Caspase-3 activity increased linearly over a period of 24-hour stimulation (p<.01), but this augmentation was blunted by 50% after DFMO administration (p<.05). The effect of DFMO on TNFalpha/MG132-induced upregulation of caspase-3 activity was reversed by the addition of 100 microM putrescine, confirming that polyamines were really involved in the apoptotic process. Also, the number of apoptotic BMSCs after TNFalpha/MG132 treatment, as determined by terminal transferase-mediated dUTP nick end-labeling (TUNEL) assay, were threefold reduced after polyamine depletion (p<.05). On the contrary, DFMO did not affect the MG132-mediated increase in p53 abundance, nor its translocation to the nucleus. Thus, polyamine depletion can be considered a useful tool for counteracting programmed cell death in BMSCs without involving the p53 proapoptotic protein.

The concomitant presence of polymorphic alleles of interleukin-1beta, interleukin-6 and apolipoprotein E is associated with an increased risk of myocardial infarction in elderly men. Results from a pilot study.

Genetic background of inflammatory or anti-inflammatory molecules may be helpful in identifying subjects with increased or decrease risk of developing cardiovascular disease. Bi-allele polymorphism (C > T) in the promoter region (-511) of the interleukin-1beta (IL-1beta) gene and the bi-allele polymorphism (G > C) in the promoter region (-174) of interleukin-6 (IL-6) gene were determined in elderly men patients with myocardial infarction (MI) and healthy controls. Each subject was also genotyped for the triallelic polymorphism of the apolipoprotein E epsilon gene. The IL-6C and APOE epsilon4 alleles were independently associated with a mild or moderate increased risk of MI, whilst the allele C of the IL-1beta was not independently linked to MI risk. However, the simultaneous presence of the allele C of IL-1beta, the allele C of IL-6 and epsilon4 allele of APOE was strongly associated with the disease. Data from this cross-sectional study suggest that the functional interaction of these three genes affects pathogenetic mechanisms of MI and an impaired regulation of immune responses plays a pivotal role in the disease. Furthermore, genetic background of inflammatory genes may influence longevity of human species by affecting inflammatory responses associated to cardiovascular diseases. The administration of anti-inflammatory compounds to middle age healthy subjects with increased genetic susceptibility of developing MI might decrease the incidence and prevalence of cardiovascular events in aging.

Evaluation of cellular energetics by the pasteur effect in intact cardiomyoblasts and isolated perfused hearts.

This work aims at exploring changes in cellular energetics by exploiting the Pasteur effect. We assumed that lactate overproduction arising from antimycin A-induced inhibition of mitochondrial respiration (delta-lactate = stimulated [lactate] -basal [lactate]) is indicative of the energy provided aerobically by the cell. Rat embryonal cardiomyocytes (H9c2), incubated with 2 micromol/L antimycin A, increased about 6 fold their lactate production in a manner linear with time and cell number. Antimycin A was also delivered to Langendorff-perfused rat hearts under control aerobic conditions or after 20 min-ischemia and 30 min-reperfusion. The test started at the end of each perfusion and lactate was measured into perfusate collected for further 25 min. A cardioplegic solution was also delivered during the test to exclude that lactate production was influenced by cardiac contraction. Control delta-lactate was 20.9 +/- 2.31 (S.E.M.) microg/mL and markedly decreased after reperfusion (7.66 +/- 0.51, p < 0.001), showing that energy production was impaired of about 70%. The determination of oxygen consumption by mitochondria isolated from reperfused hearts also suggested that the damage to the respiratory chain was similar to that evaluated by lactate overproduction (Respiratory Control Index: 75% lower than control, p < 0.001). Moreover, when delta-lactate was referred to the estimated cells which remained viable at the end of reperfusion (49.9%), it was 25% lower than control (p < 0.05). Therefore, we proposed this test as a tool for quantifying both physiological and pathological energetic modifications in living intact cardiomyocytes and in isolated and perfused hearts.

Early preconditioning prevents the loss of endothelial nitric oxide synthase and enhances its activity in the ischemic/reperfused rat heart.

Cardiac ischemia may be responsible for either the loss of endothelial nitric oxide synthase (eNOS) or changes in its activity, both conditions leading to coronary dysfunction. We investigated whether early ischemic preconditioning was able to preserve eNOS protein expression and function in the ischemic/reperfused myocardium. Langendorff-perfused rat hearts were subjected to 20 min global ischemia, followed by 30 min reperfusion (I/R). A second group of hearts was treated as I/R, but preconditioned with three cycles of 5 min-ischemia/5 min-reperfusion (IP). Cardiac contractility markedly decreased in I/R, consistently with the rise of creatine kinase (CK) activity in the coronary effluent, whilst ischemic preconditioning significantly improved all functional parameters and reduced the release of CK. Western blot analysis revealed that the amount of eNOS protein decreased by 54.2% in I/R with respect to control (p < 0.01). On the other hand, NOS activity was not significantly reduced in I/R, as well as cGMP tissue levels, suggesting that a parallel compensatory stimulation of this enzymatic activity occurred during ischemia/reperfusion. Ischemic preconditioning completely prevented the loss of eNOS. Moreover, both NOS activity and cGMP tissue level were significantly higher (p < 0.05) in IP (12.7 +/- 0.93 pmol/min/mg prot and 58.1 +/- 12.2 fmol/mg prot, respectively) than I/R (7.34 +/- 2.01 pmol/min/mg prot and 21.4 +/- 4.13 fmol/mg prot, respectively). This suggest that early ischemic preconditioning may be useful to accelerate the complete recovery of endothelial function by preserving the level of cardiac eNOS and stimulating the basal production of nitric oxide.